The overarching goal of this project is to understand the pathogenesis of Rett syndrome (RTT), a neurodevelopmental disorder and a major cause of mental retardation in females. Mutations in the X-linked gene encoding methyl-CpG- binding protein 2 (MeCP2) cause RTT. MeCP2 binds to methylated cytokines at CpG-dinucleotides, which leads to chromatin changes and transcriptional repression. We propose that the predominantly neurological RTT phenotype results from the differential expression of MECP2 variants, which affects the stability and/or abundance of the gene product in the central nervous system (CNS), and that RTT is caused by defective methylation-dependently silencing of genes relevant to neurodevelopment. To test these hypotheses, we will carry out cell biological and molecular studies using both patient and animal tissues. We will determine the expression of various alternative RNA transcripts of MECP2 in different regions of the CNS during development and evaluate the distribution of MeCP2 protein in neuronal versus peripheral tissues of healthy controls and RTT patients. We will determine how MECP2 mutations affect gene expression and early development by studying: (a) targeted mouse embryonic stem cells during in vitro differentiation, (b) altered gene expression in Xenopus embryos after over-expression of mutated human synthetic MECP2- RNA, and (c) altered gene expression in cell lines from RTT patients. We will also assess the therapeutic effects of the methyl donors folate and betaine on DNA methylation and gene expression when administered to mice with MECP2 mutations. This work will provide substantive insights into the pathology of RTT and the role of DNA methylation in development of the nervous system. It will allow us to identify and further characterize downstream genes that are responsible for the very specific features of RTT. RTT may define a novel class of inherited disorders caused by methylation-dependent imbalances of epigenetic regulation and the findings of this study may therefore by applicable to a variety of other conditions. For example, we may better understand how folate prevents neural tube defects. Finally, these findings may lead to a therapeutic strategy for RTT.